Two-photon FCS System

描述(申请人提供)：我们申请资金购买共享的双光子荧光相关光谱(PCS)系统。该系统最初将有四个主要用户，所有这些用户都有资格资助的NIH项目，其中特别包括使用PCS。这些项目都需要量化蛋白质的相互作用，涵盖一系列系统，包括激酶、转运蛋白和伴侣蛋白。这些资助项目的初步数据是使用安装的蔡司LSM 510 Conafor II获得的，但这一现有仪器有两个主要限制。首先，它在安妮·肯沃西博士的实验室里，被广泛用于她所有的共焦和PCS项目。Conafor II只能通过合作协议提供给其他实验室，而不是作为服务费提供。随着肯沃西实验室的发展，我们需要的PCS实验将很难访问这个系统。其次，ConFocor II仅限于共焦检测，不能用于双光子激发(因为它已经安装了405 nm激光选项)。如本提案所述，双光子激发便于数据分析和解释，因为非线性激发产生了明确的焦体积。 拟议的仪器将成为细胞成像共享资源的一部分。所有主要用户都可以通过该资源访问该仪器和培训。此外，新用户也将能够使用该仪器。该资源在范德比尔特大学拥有220多个实验室小组，在教育、培训和生产力方面有着广泛的记录。在过去的11年里，我们引入了共用共焦显微镜、活细胞成像、双光子激发、全内反射(TIRF)显微镜和去卷积显微镜。这些技术最初被更多的生物物理实验室使用，但很快就被普通生物医学研究界广泛使用。我们预计未来几年PCS的使用也会出现类似的扩大。 我们选择ISS ALBA仪器是因为它的灵活性，允许不同的激光进行激发和多种分析模式。特别是，我们需要光子计数直方图(PCH)分析来从分子相互作用实验中提取最大数量的信息。传统的自相关分析通过扩散系数来区分物种(很难区分单体和二聚体)，而PCH分析则是基于分子亮度来区分物种。这一分析将极大地促进我们所有正在进行的项目。最后，开放的ALBA数据结构允许在开发其他分析方法时应用它们。

DESCRIPTION (provided by applicant): We request funds to purchase a shared two-photon fluorescence correlation spectroscopy (PCS) system. This system will have four initial major users, all of whom have qualifying funded NIH projects that specifically include the use of PCS. These projects all require quantitation of protein interactions and cover a range of systems including kinases, transporters, and chaperones. Preliminary data for these funded projects was acquired using the installed Zeiss LSM 510 Confocor II, but this existing instrument has two major limitations. First, it is in the laboratory of Dr. Anne Kenworthy, and is used extensively for all of her confocal and PCS projects. The Confocor II is available to other labs only through collaborative agreements and not as fee-for-service. As the Kenworthy lab grows, this system will be difficult to access for our needed PCS experiments. Second, the Confocor II is limited to confocal detection, and cannot be adapted for two-photon excitation (because it already has the 405 nm laser option installed). As described in this proposal, two-photon excitation facilitates data analysis and interpretation, because of the well-defined focal volume generated by the non-linear excitation. The proposed instrument will be part of the Cell Imaging Shared Resource. All major users will have access to the instrument and training through the Resource. In addition, new users will also be able to use this instrument. The Resource has an extensive track record of education, training, and productivity with over 220 lab groups at Vanderbilt University. Over the last 11 years, we have introduced shared access to confocal microscopy, live cell imaging, two photon excitation, total internal reflection (TIRF) microscopy, and deconvolution microscopy. These techniques began with use by the more biophysical laboratories, but rapidly became widely used by the general biomedical research community. We foresee a similar broadening in PCS use over the next few years. We have chosen the ISS Alba instrument because of its flexibility, which allows different lasers for excitation and multiple analysis modes. In particular, we require the Photon Counting Histogram (PCH) analysis to extract the maximal amount of information from molecular interaction experiments. While conventional autocorrelation analysis separates species by diffusion coefficient (making it difficult to distinguish between monomers and dimers), the PCH analysis separates species based on molecular brightness. This analysis will greatly facilitate all of our on-going projects. Finally, the open Alba data structure allows other analysis methods to be applied as they are developed.